This invention relates to nucleic acid molecules and encoded polypeptides which are expressed preferentially in tumors. The nucleic acid molecules and encoded polypeptides are useful in, inter alia, diagnostic and therapeutic contexts.
The phenotypic changes which distinguish a tumor cell from its normal counterpart are often the result of one or more changes to the genome of the cell. The genes which are expressed in tumor cells, but not in normal counterparts, can be termed xe2x80x9ctumor associatedxe2x80x9d genes. These tumor associated genes are markers for the tumor phenotype. The expression of tumor associated genes can also be an essential event in the process of tumorigenesis.
Typically, the host recognizes as foreign the tumor associated genes which are not expressed in normal non-tumorigenic cells. Thus, the expression of tumor associated genes can provoke an immune response against the tumor cells by the host. Tumor associated genes can also be expressed in normal cells within certain tissues without provoking an immune response. In such tissues, expression of the gene and/or presentation of an ordinarily immunologically recognizable fragment of the protein product on the cell surface may not provoke an immune response because the immune system does not xe2x80x9cseexe2x80x9d the cells inside these immunologically privileged tissues. Examples of immunologically privileged tissues include brain and testis
The discovery of tumor associated expression of a gene provides a means of identifying a cell as a tumor cell. Diagnostic compounds can be based on the tumor associated gene, and used to determine the presence and location of tumor cells. Further, when the tumor associated gene is essential for an aspect of the tumor phenotype (e.g., unregulated growth or metastasis), the tumor associated gene can be used to provide therapeutics such as antisense nucleic acids which can reduce or substantially eliminate expression of that gene, thereby reducing or substantially eliminating the phenotypic aspect which depends on the expression of the particular tumor associated gene.
As previously noted, the polypeptide products of tumor associated genes can be the targets for host immune surveillance and provoke selection and expansion of one or more clones of cytotoxic T lymphocytes specific for the tumor associated gene product. Examples of this phenomenon include proteins and fragments thereof encoded by the MAGE family of genes, the tyrosinase gene, the Melan-A gene, the BAGE gene, the GAGE gene, the RAGE family of genes, the PRAME gene and the brain glycogen phosphorylase gene, as are detailed below. Thus, tumor associated expression of genes suggests that such genes can encode proteins which will be recognized by the immune system as foreign and thus provide a target for tumor rejection. Such genes encode xe2x80x9ctumor rejection antigen precursorsxe2x80x9d, or TRAPs, which may be used to generate therapeutics for enhancement of the immune system response to tumors expressing such genes and proteins.
The process by which the mammalian immune system recognizes and reacts to foreign or alien materials is a complex one. An important facet of the system is the T cell response. This response requires that T cells recognize and interact with complexes of cell surface molecules, referred to as human leukocyte antigens (xe2x80x9cHLAxe2x80x9d), or major histocompatibility complexes (xe2x80x9cMHCsxe2x80x9d), and peptides. The peptides are derived from larger molecules which are processed by the cells which also present the HLA/MHC molecule. See in this regard Male et al., Advanced Immunology (J. P. Lipincott Company, 1987), especially chapters 6-10. The interaction of T cells and complexes of HLA/peptide is restricted, requiring a T cell specific for a particular combination of an HLA molecule and a peptide. If a specific T cell is not present, there is no T cell response even if its partner complex is present. Similarly, there is no response if the specific complex is absent, but the T cell is present. The mechanism is involved in the immune system""s response to foreign materials, in autoimmune pathologies, and in responses to cellular abnormalities. Much work has focused on the mechanisms by which proteins are processed into the HLA binding peptides. See, in this regard, Barinaga, Science 257:880, 1992; Fremont et al., Science 257:919, 1992; Matsumura et al., Science 257:927, 1992; Latron et al., Science 257:964, 1992.
The mechanism by which T cells recognize cellular abnormalities has also been implicated in cancer. For example, in PCT application PCT/US92/04354, filed May 22, 1992, published on Nov. 26, 1992, and incorporated by reference, a family of genes is disclosed, which are processed into peptides which, in turn, are expressed on cell surfaces, which can lead to lysis of the tumor cells by specific CTLs The genes are said to code for xe2x80x9ctumor rejection antigen precursorsxe2x80x9d or xe2x80x9cTRAPxe2x80x9d molecules, and the peptides derived therefrom are referred to as xe2x80x9ctumor rejection antigensxe2x80x9d or xe2x80x9cTRAsxe2x80x9d. See Traversari et al., J. Exp. Med. 176:1453-1457, 1992; van der Bruggen et al., Science 254:1643, 1991; De Plaen et al., Immunogenetics 40:360-369, 1994 for further information on this family of genes. Also, see U.S. patent application Ser. No. 807,043, filed Dec. 12, 1991, now U.S. Pat. No. 5,342,774.
In U.S. patent application Ser. No. 938,334, now U.S. Pat. No. 5,405,940, the disclosure of which is incorporated by reference, nonapeptides are taught which are presented by the HLA-A1 molecule. The reference teaches that given the known specificity of particular peptides for particular HLA molecules, one should expect a particular peptide to bind one HLA molecule, but not others. This is important, because different individuals possess different HLA phenotypes. As a result, while identification of a particular peptide as being a partner for a specific HLA molecule has diagnostic and therapeutic ramifications, these are only relevant for individuals with that particular HLA phenotype. There is a need for further work in the area, because cellular abnormalities are not restricted to one particular HLA phenotype, and targeted therapy requires some knowledge of the phenotype of the abnormal cells at issue.
In U.S. patent application Ser. No. 008,446, filed Jan. 22, 1993 and incorporated by reference, the fact that the MAGE-1 expression product is processed to a second TRA is disclosed. This second TRA is presented by HLA-Cw16 molecules, also known as HLA-C*1601. The disclosure shows that a given TRAP can yield a plurality of TRAs.
In U.S. patent application Ser. No. 994,928, filed Dec. 22, 1992, and incorporated by reference herein, tyrosinase is described as a tumor rejection antigen precursor. This reference discloses that a molecule which is produced by some normal cells (e.g., melanocytes), is processed in tumor cells to yield a tumor rejection antigen that is presented by HLA-A2 molecules.
In U.S. patent application Ser. No. 08/032,978, filed Mar. 18, 1993, and incorporated herein by reference in its entirety, a second TRA, not derived from tyrosinase is taught to be presented by HLA-A2 molecules. The TRA is derived from a TRAP, but is coded for by a known MAGE gene. This disclosure shows that a particular HLA molecule may present TRAs derived from different sources.
In U.S. patent application Ser. No. 079,110, filed Jun. 17, 1993 and entitled xe2x80x9cIsolated Nucleic Acid Molecules Coding For BAGE Tumor Rejection Antigen Precursorsxe2x80x9d and Ser. No. 196,630, filed Feb. 15, 1994, and entitled xe2x80x9cIsolated Peptides Which form Complexes with MHC Molecule HLA-C-Clone 10 and Uses Thereofxe2x80x9d the entire disclosures of which are incorporated herein by reference, an unrelated tumor rejection antigen precursor, the so-called xe2x80x9cBAGExe2x80x9d precursor, is described. TRAs are derived from the TRAP and also are described. They form complexes with MHC molecule HLA-C-Clone 10.
In U.S. patent application Ser. No. 096,039, filed Jul. 22, 1993 and entitled xe2x80x9cIsolated Nucleic Acid Molecules Coding for GAGE Tumor Rejection Antigen Precursorsxe2x80x9d and Ser. No. 250,162, filed May 27, 1994 and entitled xe2x80x9cMethod for Diagnosing a Disorder by Determining Expression of GAGE Tumor Rejection Antigen Precursorsxe2x80x9d, the entire disclosures of which are incorporated herein by reference, another unrelated tumor rejection antigen precursor, the so-called xe2x80x9cGAGExe2x80x9d precursor, is described. The GAGE precursor is not related to the BAGE or the MAGE family.
In U.S. patent application Ser. No. 08/408,015, filed Mar. 21, 1995, and entitled xe2x80x9cRAGE Tumor Rejection Antigen Precursorsxe2x80x9d, incorporated herein by reference in its entirety, another TRAP is taught which is not derived from any of the foregoing genes. The TRAP is referred to as RAGE. In U.S. patent application Ser. No. 08/530,015, filed Sep. 20, 1995, and entitled xe2x80x9cIsolated RAGE-1 Derived Peptides Which Complex with HLA-B7 Molecules and Uses Thereofxe2x80x9d, also incorporated by reference, the TRA derived form one member of the RAGE family of genes is taught to be presented by HLA-B7 molecules. This disclosure shows that additional TRAPs and TRAs can be derived from different sources.
In U.S. patent application Ser. No. 08/253,503, filed Jun. 3, 1994, and entitled xe2x80x9cIsolated Nucleic Acid Molecule Which Codes for a Tumor Rejection Antigen Precursor Which is Processed to an Antigen Presented by HLA-B44xe2x80x9d, incorporated herein by reference in its entirety, another TRAP is taught which is not derived from any of the foregoing genes. The gene encoding the TRAP is referred to as MUM-1. A tumor rejection antigen, LB-33B, is described in the application.
In U.S. patent application Ser. No. 08/373,636, filed Jan. 17, 1995, and entitled xe2x80x9cIsolated Nucleic Acid Molecule Which Codes for a Tumor Rejection Antigen Precursor Which is Processed to Antigens Presented by HLA Molecules and Uses Thereofxe2x80x9d, incorporated herein by reference in its entirety, other TRAPs are taught which are derived from LB33 and presented by HLA-B13, HLA-Cw6, HLA-A28 and HLA-A24.
In PCT publication WO96/10577, published Apr. 11, 1996, and entitled xe2x80x9cIsolated Nucleic Acid Molecule Coding for a Tumor Rejection Antigen Precursor DAGE and Uses Thereofxe2x80x9d, incorporated herein by reference in its entirety, another TRAP is taught which is not derived from any of the foregoing genes. The TRAP was referred to as DAGE, but is now referred to as PRAME. A tumor rejection antigen is described in the application which is presented by HLA-A24.
In U.S. patent application Ser. No. 08/487,135, filed Jun. 7, 1995, and entitled xe2x80x9cIsolated Nucleic Acid Molecule, Peptides Which Form Complexes with MHC Molecule HLA-A2 and Uses Thereofxe2x80x9d, incorporated herein by reference in its entirety, another TRAP is taught which is not derived from any of the foregoing genes. The TRAP is referred to as NAG. Various TRAs derived from NAG and presented by HLA-A2 are taught in this application.
In U.S. patent application Ser. No. 08/403,388, filed Mar. 14, 1995, and entitled xe2x80x9cIsolated Nucleic Acid Molecules Which Are Members of the MAGE-Xp Family and Uses Thereofxe2x80x9d, incorporated herein by reference in its entirety, three TRAPs are taught which are not derived from any of the foregoing genes. These TRAPs are referred to as MAGE-Xp2, MAGE-Xp3 and MAGE-Xp4.
The work which is presented by the papers, patents and patent applications described above deal, for the most part, with the MAGE family of genes, the BAGE gene, the GAGE gene and the RAGE family of genes. It now has been discovered that additional genes similarly are expressed in a tumor associated pattern.
The invention is elaborated upon further in the disclosure which follows.
The genes which are believed to encode tumor rejection antigen precursors were referred to originally as LL-1 tumor associated genes (LL-1.1 and LL-1.2). One of the two genes, originally termed LL-1.2, is now known as NY-ESO-1 as described in U.S. patent application Ser. No. 08/725,182. The other LL-1 gene, LL-1.1, is now known as LAGE-1 and does not show homology to the MAGE family of genes, to the BAGE gene, the GAGE gene, the RAGE family of genes, the LB33/MUM-1 gene, the PRAME gene, the NAG gene or the MAGE-Xp family of genes. Thus the invention relates to the LAGE-1 gene expressed specifically in certain tumor cells, tumor rejection antigen precursors encoded by the LAGE-1 gene, as well as related molecules and applications of these various entities.
The invention provides isolated nucleic acid molecules, unique fragments of those molecules, expression vectors containing the foregoing, and host cells transfected with those molecules. The invention also provides isolated polypeptides and agents which bind such polypeptides, including antibodies. Kits for detecting the presence of a LAGE-1 tumor associated polypeptide precursor additionally are provided. The foregoing can be used in the diagnosis or treatment of conditions characterized by the expression of a LAGE-1 tumor-specific polypeptide or precursor thereof.
According to one aspect of the invention, an isolated nucleic acid molecule is provided. The molecule hybridizes under stringent conditions to a molecule having a nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID NO:4 and the nucleotide sequence of SEQ ID NO:6. The isolated nucleic acid molecule is a LAGE-1 tumor associated polypeptide precursor and codes for a LAGE-1 tumor associated polypeptide, including allelic variants of LAGE-1 tumor associated polypeptides. The invention further embraces nucleic acid molecules that differ from the foregoing isolated nucleic acid molecules in codon sequence to the degeneracy of the genetic code. The invention also embraces complements of the foregoing nucleic acids.
In preferred embodiments, the isolated nucleic acid molecule comprises a molecule selected from the group consisting of the nucleic acid sequence of SEQ ID NO:4 and the nucleic acid sequence of SEQ ID NO:6. More preferably, the isolated nucleic acid molecule comprises a molecule selected from the group consisting of the coding region of the nucleic acid sequence of SEQ ID. NO:4 and the coding region of the nucleic acid sequence of SEQ ID NO:6.
According to another aspect of the invention, an isolated nucleic acid molecule is provided which comprises a molecule selected from the group consisting of a unique fragment of nucleotides 1-993 of SEQ ID NO:4 between 12 and 992 nucleotides in length, a unique fragment of nucleotides 1-746 of SEQ ID NO:6 between 12 and 745 nucleotides in length, and complements thereof. The unique fragments exclude nucleic acid molecules which consist only of fragments of SEQ ID NO:8 and fragments of SEQ ID NO:8 having 5 or fewer contiguous nucleotides of SEQ ID NO:4 OR SEQ ID NO:6. In preferred embodiments, the unique fragment is at least 14, 15, 16, 17, 18, 20 or 22 contiguous nucleotides of nucleotides 1-993 of SEQ ID NO:4, nucleotides 1-746 SEQ ID NO:6, or complements thereof. In another embodiment, the isolated nucleic acid molecule consists of between 12 and 32 contiguous nucleotides of nucleotides 1-993 of SEQ ID NO:4, nucleotides 1-746 of SEQ ID NO:6, or complements of such nucleic acid molecules.
According to another aspect of the invention, the invention involves expression vectors, and host cells transformed or transfected with such expression vectors, comprising the nucleic acid molecules described above. The expression vectors and/or host cells preferably include a nucleic acid molecule which codes for a HLA molecule. Of course, an HLA-encoding nucleic acid molecule can also be contained in a separate expression vector.
According to another aspect of the invention, an isolated polypeptide encoded by a nucleic acid molecule which hybridizes under stringent conditions to a molecule selected from the group consisting of the nucleic acid sequence of SEQ ID NO:4 and the nucleic acid sequence of SEQ ID NO:6, nucleic acid molecules which vary from the foregoing according to the degeneracy of the genetic code, complements and allelic variants of any of the foregoing nucleic acid molecules. Preferred polypeptides are those which include the amino acid sequence of SEQ ID NO:5, the amino acid sequence of SEQ ID NO:7, the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:7 having a glutamine to arginine substitution at residue 6, the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:7 having a glutamine to glutamic acid substitution at residue 89, and the amino acid sequence of SEQ ID NO:5 having an arginine to tryptophan substitution at residue 138.
In another aspect of the invention, isolated LAGE-1 polypeptides which include amino acids 89-93 of SEQ ID NO:5 or 7 are provided. Preferred embodiments of such polypeptides include isolated LAGE-1 polypeptides which include amino acids 71-93, 71-98, 89-98, 89-111, or 71-111 of SEQ ID NO:5 or 7. Nucleic acids which encode such polypeptides also are provided.
According to another aspect of the invention, isolated LAGE-1b polypeptides which include amino acids 142-148 of SEQ ID NO:5, amino acids 187-205 of SEQ ID NO:5, or amino acids 164-179 of SEQ ID NO:5 are provided. Preferably such isolated polypeptides include amino acids 134-210 of SEQ ID NO:5. Isolated nucleic acids which encode such polypeptides also are provided.
According to yet another aspect of the invention, an isolated polypeptide is provided which comprises a molecule selected from the group consisting of a unique fragment of SEQ ID NO:5 between 9 and 209 amino acids in length and a unique fragment of SEQ ID NO:7 between 9 and 179 amino acids in length. The unique fragment is not a polypeptide consisting of fragments of SEQ ID NO:9. Preferably, the unique fragment of the isolated polypeptide binds to a polypeptide-binding agent. In preferred embodiments, the polypeptide-binding agent is an antibody or a cytotoxic T lymphocyte.
The invention also provides isolated polypeptides which selectively bind a LAGE-1 protein or fragments thereof. Isolated binding polypeptides include antibodies and fragments of antibodies (e.g., Fab, F(ab)2, Fd and antibody fragments which include a CDR III region which binds selectively to the LAGE-1 proteins of the invention). The isolated binding polypeptides include monoclonal antibodies.
The invention in another aspect involves a kit for detecting the presence of the expression of a LAGE-1 tumor associated polypeptide precursor. Such kits employ two or more of the above-described molecules isolated in separate containers and packaged in a single package. In one such kit, a pair of isolated nucleic acid molecules is provided, each of the pair consisting essentially of a molecule selected from the group consisting of a 12-32 nucleotide contiguous segment of SEQ ID NO:4 and complements thereof, and a 12-32 nucleotide contiguous segment of SEQ ID NO:6 and complements thereof, wherein the contiguous segments are nonoverlapping. Preferably, the pair of isolated nucleic acid molecules is constructed and arranged to selectively amplify an isolated nucleic acid molecule which hybridizes under stringent conditions to a molecule selected from the group consisting of the nucleic acid sequence of SEQ ID NO:4, the nucleic acid sequence of SEQ ID NO:6, nucleic acid molecules which differ from the above in codon sequence due to the degeneracy of the genetic code, complements and allelic variants thereof. In certain embodiments, the pair of isolated nucleic acid molecules is PCR primers. Preferably one of the primers is a contiguous segment of SEQ ID NO:4 and another of the primers is a complement of another contiguous segment of SEQ ID NO:4. In other preferred embodiments, one of the primers is a contiguous segment of SEQ ID NO:6 and another of the primers is the complement of another contiguous segment of SEQ ID NO:6.
According to still another aspect of the invention, a method for diagnosing a disorder characterized by the expression of a LAGE-1 nucleic acid molecule or an expression product thereof is provided. The method involves contacting a biological sample isolated from a subject with an agent that selectively binds a LAGE-1 nucleic acid molecule or an expression product thereof. In certain embodiments, the nucleic acid molecule hybridizes under stringent conditions to a molecule selected from the group consisting of the nucleic acid sequence of SEQ ID NO:4 and the nucleic acid sequence of SEQ ID NO:6, and which codes for a tumor associated polypeptide. In other embodiments, the agent is a binding agent which selectively binds to a LAGE-1 tumor associated polypeptide, such as an antibody, cytotoxic T lymphocyte, polypeptide, and the like. The method further involves determining the interaction between the agent and the nucleic acid molecule or expression product thereof as a determination of the disorder. In preferred embodiments, the agent is a DNA molecule comprising SEQ ID NO:4 or SEQ ID NO:6, or a unique fragment thereof. In certain embodiments, the interaction between the agent and the nucleic acid molecule is determined by amplifying at least a portion of the nucleic acid molecule.
According to another aspect of the invention, a method for treating a subject with a disorder characterized by expression of an LAGE-1 tumor associated polypeptide is provided. The method involves administering to the subject an amount of an agent, which agent enriches selectively in the subject the presence of complexes of a HLA molecule and a tumor rejection antigen which is derived from a LAGE-1 tumor associated polypeptide coded for by one of the foregoing nucleic acid molecules. The amount of the agent administered in sufficient to ameliorate the disorder. Preferably the agent is a LAGE-1 polypeptide, or an immunogenic fragment thereof.
According to yet another aspect of the invention, a method for treating a subject with a disorder characterized by expression of a LAGE-1 nucleic acid molecule or an expression product thereof is provided. The method includes administering to the subject an amount of autologous cytolytic T cells sufficient to ameliorate the disorder, wherein the cytolytic T cells are specific for complexes of an HLA molecule and a LAGE-1 tumor associated polypeptide or an immunogenic fragment thereof.
In another aspect, the invention provides methods for treating a subject with a disorder characterized by expression of a LAGE-1 nucleic acid molecule or an expression product thereof. The methods include administering to the subject an amount of a LAGE-1 tumor associated polypeptide or an immunogenic fragment thereof sufficient to ameliorate the disorder.
According to another aspect of the invention, methods for enriching selectively a population of T cells with cytolytic T cells specific for a LAGE-1 tumor associated polypeptide are provided. The methods include contacting an isolated population of T cells with an agent presenting a complex of a LAGE-1 tumor associated polypeptide or an immunogenic fragment thereof and a HLA presenting molecule in an amount sufficient to selectively enrich the isolated population of T cells with the cytolytic T cells. Preferably the agent is a cell which expresses a LAGE-1 tumor associated polypeptide and a HLA molecule. In certain preferred embodiments, the LAGE-1 tumor associated polypeptide is encoded by a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:4 or SEQ ID NO:6.
According to other aspects of the invention, vaccine compositions which include a nucleic acid encoding at least one LAGE-1 epitope, a LAGE-1 polypeptide and/or a cell which expresses LAGE-1 nucleic acid or polypeptide, or immunogenic fragments thereof, are provided. The vaccine compositions are useful for increasing an immune response in a subject.
Use of the foregoing compositions in the preparation of medicaments is also provided. In particular, use of the compositions in the preparation of medicaments is also provided. In particular, use of the composition in the preparation of a medicament for treating cancer is provided.
These and other objects of the invention will be described in further detail in connection with the detailed description of the invention.